Project Summary Covalent modifications of histones, such as acetylation, methylation, phosphorylation, and ubiquitylation, are essential regulators of chromatin structure and function. Defects in the regulation of these modifications have causal roles in numerous developmental disorders and diseases. However, the mechanisms that target histone-modifying enzymes to specific genomic locations and regulate their enzymatic activities are not well understood. Our long-term goal is to understand how diverse histone modification activities are coordinated to initiate and maintain different epigenetic states using heterochromatin assembly and oncogenic histone mutations as experimental models. Heterochromatin preferentially assembles at repetitive DNA elements and it is essential for the regulation of gene expression and the maintenance of genome integrity. Formation of heterochromatin is critically dependent on the methylation of H3 lysine 9 (H3K9), and it is generally assumed that precise targeting of histone H3K9 methyltransferases confines heterochromatin to specific genomic regions. However, our recent studies demonstrate that in fission yeast the targeting of H3K9 methyltransferases Clr4 is not very precise, and cells rely critically on negatively regulators, such as the Mst2 histone acetyltransferase and the Epe1 histone demethylase, to remove heterochromatin at inappropriate locations. We will therefore analyze the molecular functions of Mst2 and Epe1 in heterochromatin formation, and examine how their activities change in response to environmental signals to regulate heterochromatin dynamics. Recent high throughput sequencing analyses discovered high incidences of somatic histone lysine-to- methionine (K-to-M) mutations in multiple cancers. These mutations block the methylation of wild type histones. However, the molecular details by which these mutations function are poorly understood and are highly controversial. We have established fission yeast models in which the introduction of H3K9M or H3K36M transgenes abolished the methylation of corresponding lysines on wild type histones, similar to the effects of these mutations in mammalian systems. We will examine how these mutations regulate cellular functions and identify pathways that can be targeted to selectively kill cells containing K-to-M mutations. The ultimate goal of these studies is a complete understanding of how histone methylations are regulated and how their mutations and dysregulation contribute to human diseases.